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The mutagenic/genotoxic potential of DHA was investigated in an extensive battery of studies including in vitro tests (bacterial reverse mutation assays, mammalian cell mutation test in Chinese Hamster V79 cells (hprt locus) and chromosomal aberration test in Chinese Hamster V79 cells) and an in vivo assay (bone marrow micronucleus test in mice). The robustness of this test battery was evaluated as discussed below.

(1) Bacterial reverse mutation tests

DHA was evaluated for bacterial mutagenicity in several different bacterial reverse mutation tests. Initially, two tests were carried out on two different batches of DHA. Each test comprised of two independent experiments in the presence and absence of Aroclor-induced rat S9-mix. For both tests, negative results were obtained with the strains TA98, TA1535 and TA1537 up to a maximum concentration of 10000 µg/plate, equivalent to 111 mMol/plate. In both studies, reproducible and dose-related increases in revertant counts were observed at high test concentrations ( 2500 µg/plate, i.e. 28 mMol/plate) with the test strains TA100 and TA102 in the presence and absence of S9-mix. The presence of S9-mix had no modifying effects in these tests. These results were confirmed in a third bacterial reverse mutation test performed with test strain TA100 and different modifications to the in vitro metabolizing system, i.e. different concentrations of Aroclor-induced rat S9-mix, inactivated Aroclor-induced rat S9-mix, BSA or combinations of these proteins. It was shown that the use of different amounts of S9-mix and the other proteins specified had no significant effect on the quantitative response of TA100. A fourth bacterial reverse mutation test was carried out in order to investigate a possible role for photo-activation on the observed mutagenic activity. In this test, DHA was exposed to UV-light before testing. Essentially the same results were obtained as in the previous tests: negative results were obtained with strains TA98, TA1535, and TA1537, whilst increases in revertant counts were obtained with test strains TA100 and TA102 in the presence and absence of S9-mix at high concentrations. A possible photomutagenic potential of dihydroxyacetone was investigated in a Photoames assay using the four bacterial strains S. typhimurium TA102, TA1537, TA100 and E. coli WP2 in the absence of a mammalian metabolizing system. No mutagenicity was observed in strains TA1537 and WP2 either with or without UV irradiation. As expected, DHA increased the number of revertant counts of TA100 and TA102 in the absence of UV irradiation. The magnitude of the mutagenic effects was not influenced by exposure to UV-light. The results from these studies are consistent with the lack of UV absorption shown by DHA in the UVA/UVB range of wavelength (290-400 nm). Based on all these studies it is concluded that DHA is a weak direct acting base-substitution mutagen in S. typhimurium strains TA100 and TA102. The mutagenic effects are not modified by the presence of Aroclor-induced rat S9-mix and there is no detectable photo-activation after exposure to UV-light.

(2) Other in vitro genetic toxicitity tests

The genotoxic potential of dihydroxyacetone to mammalian cells was evaluated in an hprt gene mutation assay using Chinese Hamster V79 cell. Cells were exposed in two independent experiments at test concentrations ranging from 31.6 to 1000 µg/mL (i.e. 0.3 to 11 mM) for 24 hrs in the absence of Aroclor-induced S9-mix, and concentrations ranging from 158 to 5000 µg/mL (i.e. 1.8 to 56 mM) for 2 hrs in the presence of S9. No significant increases in mutation frequencies were observed in cells exposed to various concentrations of DHA in the absence or presence of a metabolic activation system. Dihydroxyacetone was not mutagenic at the hprt locus in Chinese Hamster V79 cells. It should be noted that in the main study, no clear evidence of cytotoxicity was seen at the limit dose of 5000 µg/mL (56 mM). However this test concentration was in excess of that recommended in modern test guidelines, which state a maximal test concentration of 5000 µg/mL or 10 mM, whichever is the lowest. The clastogenic potential of DHA was evaluated in Chinese hamster V79-cells in the presence and absence of a mammalian metabolic activation system (S9). The cells were treated in two independent experiments with test concentrations up to 1400 and 2500 µg/mL (i.e. 16 to 28 mM), respectively. Cells were sampled for metaphase analysis at various time points after start of treatment. No increases in chromosomal aberrations or in the frequency of polyploidy were obtained in this study at any sampling time. Therefore, DHA was evaluated as not clastogenic in this in vitro chromosomal aberration assay. At 2500 µg/mL (28 mM) the level of cytotoxicity was slightly lower than recommended for this study. However, as with the previous studies the test concentrations were well in excess of the 5000 µg/mL or 10 mM limit stipulated by current test guidelines.

(3) In vivo genetic toxictity

DHA has been evaluated for its ability to induce clastogenic events and/or disruption of the mitotic spindle apparatus in vivo using the mouse bone marrow micronucleus assay. DHA was administered intraperitoneally as a single dose to groups of 6 male ddY mice at 1250, 2500, and 5000 mg/kg bw. Males were selected on the basis of a preliminary toxicity study in which no sex-related difference in toxicity was observed. The highest dose level used exceeded the current limit dose of 2000 mg/kg bw for non-toxic chemicals in this assay (OECD, 1997). Animals were killed 24 hrs after dosing and bone marrow was sampled for further analysis. There was no significant reduction in the PCE:NCE ratio at any dose level and, therefore, no indication of bone marrow cytotoxicity. No significant increases in micronucleus frequency were observed at any dose level. Plasma analyses were not carried out and, hence, direct evidence of bone marrow exposure is lacking, however, considering the route of administration (intraperitoneal), the high doses used (above the limit dose level recommended for this assay) and the physico-chemical properties of DHA (high water solubility, low molecular weight), sufficient systemic exposure thus bone marrow exposure to DHA can be assumed. Based on the results of this study, it is concluded that DHA did not induce micronuclei in the PCE of mouse bone marrow up to a maximum intraperitoneal dose of 5000 mg/kg bw. It should be noted that this study does not comply with current regulatory requirements, because the cell sample size of 1000 PCE was lower than currently recommended, and evaluation at 48-hours after treatment was not performed. However, considering the hydrophilic test compound and the very high test dose (far above the limit dose), these deviations are considered not to influence the reliability of the study.

(4) Summary/Conclusion

In conclusion, there exists an extensive battery of studies addressing the genotoxic potential of dihydroxyacetone. The overall package is considered to be robust, and shows that DHA causes base-pair substitutions at very high concentrations in bacterial cells, but is not mutagenic in mammalian in vitro test systems or in vivo.

Short description of key information:
The genetic toxicity of dihydroxyacetone was studied using an extensive battery of tests addressing the genotoxic potential. The overall package is considered to be robust, and shows that dihydroxyacetone causes base-pair substitutions at very high concentrations in bacterial cells, but is not
mutagenic in mammalian in vitro test systems or in vivo.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The negative results obtained with DHA in the in vitro genotoxicity assays using Chinese hamster V79 cells, and in vivo in the mouse bone marrow micronucleus test, indicate that the weak mutagenic effect of this chemical for bacterial cells is not likely to be relevant to any potential mutagenic or carcinogenic risk in mammalian species including humans. The absence of a mutagenic or carcinogenic potential of DHA in vivo was confirmed by the negative results observed in a lifetime skin painting study in mice. (See IUCLID5 section 7.7 - Carcinogenicity). Therefore, dihydroxyacetone must not be classified as genotoxic/mutagenic.